Collagen is the most abundant protein in the animal kingdom, accounting for one third or more of all animal proteins. Collagen is a major constituent of connective tissues of animal skin, tendons, bones, and the like. Also, animal bodies are constituted by numerous cells and collagen plays a key role as an intercellular matrix.
Up to the present, as many as about 20 types of genetically different collagen species have been discovered, and differences in properties and functions among them have been demonstrated. Different collagen species are referred to as type I, type II, or the like, and they have been distinguished from one another. It is known that common helical regions of collagen comprising repetitions of Gly-X-Y (wherein X and Y each independently represent any amino acid) are present in the centers of collagen molecules and that triple-helical structures are formed in such regions. Such helical regions of collagen have low immunogenicity and excellent biocompatibility because of the highly conserved sequences thereof among different animal species.
Up to the present, various types of collagens have been extracted from various animal tissues and extensively utilized in the fields of medicine, pharmaceutical products, biochemistry, cosmetics, food, or the like. Depending on the raw material from which collagen is to be extracted, the collagen species to be obtained varies. For example, type I collagen is mainly contained in and extracted from skin, tendons, or bones. Type II collagen is extracted from the cartilage, and type III collagen is extracted from blood vessel walls, for example. Type IV collagen that constitutes the basal membrane is obtained from the placenta or the like. It should be noted that several collagen species might be extracted together from a given raw material.
As a method for extracting collagen, a method involving treatment of materials such as animal bones or skin with an enzyme is known. In such a method, for example, pepsin is allowed to act on such materials in a dilute acetic acid solution. With the extraction method involving use of enzyme treatment, however, a non-helical region of collagen is cleaved with an enzyme. Also, proteins other than collagens that are present in tissues may be eluted from such tissues. Consequently, collagens may be disadvantageously degraded and denatured and a large quantity of proteins other than collagens may be disadvantageously contained in an extract. In order to obtain highly purified collagen, it is necessary to use adequate tissues as raw materials and to prevent contamination by other collagens as much as possible. In order to attain the inherent properties or functions of collagens, collagens that have not undergone degradation or denaturation are required.
Type IV collagen has at its N terminus the 7S domain, which has a triple-helical collagen structure but contains abundant cysteine residues, and at its C-terminus a non-helical region of collagen (NC1 domain). The 7S domain at the N terminus is capable of interacting with the 7S domain and forming a polymer tetrad. At the C terminal NC1, 2 molecules are capable of polymerization. These domains play very important roles for intermolecular interactions of type IV collagen. Unlike other collagens, the helical region of type IV collagen is known to contain 21 Gly-X-Y triplet interruptions. Because of such molecular features, it is very important to extract molecules having all of the functional domains, i.e., the NC1 domain, the 7S domain, and the helical region of collagen, without denaturation or degradation thereof.
In the past, a major raw material for type IV collagen was placenta. Placenta contains abundant collagens other than type IV collagens, such as type I and type V collagens. Accordingly, the placenta is not an optimal raw material from which to extract type IV collagen of high purity. Many methods for extracting type IV collagens are in accordance with the method of Sage et al. (J. Biol. Chem., vol. 254, No. 19, pp. 9893-9900, 1979), which involves solubilization of collagen by pepsin hydrolysis. JP Patent Publication (kokai) No. 11-171898 A (1999) discloses a technique of isolating a polymer fraction of type IV collagen. In this isolation technique, however, type IV collagen is solubilized by pepsin hydrolysis and then extracted, as with the method of Sage et al. With such techniques, type IV collagens having functional domains similar to those in vivo cannot be extracted. Further, collagens have features such that collagen molecules become bound to and aggregated with one another because of the properties of their helical regions. Accordingly, collagen cannot be purified by a technique involving the use of a column that is commonly used for protein purification. Thus, it is very difficult to remove contaminating proteins other than collagens or other collagen species at the time of extraction. Therefore, type IV collagens of high purity have not been obtained.